A long-term objective of our research is to clarify the mechanism of rhomboid intramembrane proteases. Rhomboids are integral membrane proteins and represent an ancient and widespread enzyme family. During the course of evolution rhomboids have acquired many different biological functions, many of which are important in biology and medicine. For example, in P. falciparum, the parasite that causes malaria, rhomboid proteases play an essential role in the invasion of human host cells. We have previously determined the x-ray structures of E. coli rhomboid GlpG and its complexes with mechanism-specific inhibitors. In this application we continue to use GlpG as a model system to investigate how rhomboid interacts with TM substrate. Three specific aims are proposed. In aim 1, we test the hypothesis that substrate's TM domain docks onto an exosite on GlpG and induces a conformational change that activates the protease. Crystal structures of activity- enhancing mutants and the complex between GlpG and substrate's TM domain will be solved to explain the nature of the allosteric transition. In aim 2, cysteine accessibility and optical tweezers experiments are planned to investigate the unfolding of substrate's TM domain, a prerequisite for the intramembrane cleavage reaction. In aim 3, building on the discovery of diisopropyl fluorophosphate as a covalent inhibitor for GlpG, two types of peptidomimetics incorporating reactive phosphonate groups will be synthesized to prepare stable complexes with GlpG for x-ray analysis. Mutagenesis and functional experiments are also planned to explain rhomboid's substrate specificity.